Phylogeny and Evolutionary Patterns in the Dwarf Crayfish

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Phylogeny and Evolutionary Patterns in the DwarfCrayfish Subfamily (Decapoda: Cambarellinae)C. Pedraza-Lara

I. Doadrio

J. Breinholt

Keith A. CrandallGeorge Washington University

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APA CitationPedraza-Lara, C., Doadrio, I., Breinholt, J., & Crandall, K. A. (2012). Phylogeny and Evolutionary Patterns in the Dwarf CrayfishSubfamily (Decapoda: Cambarellinae). PLoS ONE, 7 (11). http://dx.doi.org/10.1371/journal.pone.0048233

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Phylogeny and Evolutionary Patterns in the DwarfCrayfish Subfamily (Decapoda: Cambarellinae)Carlos Pedraza-Lara1,2*, Ignacio Doadrio1, Jesse W. Breinholt3, Keith A. Crandall3,4

1 Departamento de Biodiversidad y Biologıa Evolutiva, Museo Nacional de Ciencias Naturales, CSIC, Madrid, Spain, 2 Instituto de Biologıa, Universidad Nacional Autonoma

de Mexico, Distrito Federal, Mexico, 3 Department of Biology, Brigham Young University, Provo, Utah, United States of America, 4 Computational Biology Institute, George

Washington University, Ashburn, Virginia, United States of America

Abstract

The Dwarf crayfish or Cambarellinae, is a morphologically singular subfamily of decapod crustaceans that contains only onegenus, Cambarellus. Its intriguing distribution, along the river basins of the Gulf Coast of United States (Gulf Group) and intoCentral Mexico (Mexican Group), has until now lacked of satisfactory explanation. This study provides a comprehensivesampling of most of the extant species of Cambarellus and sheds light on its evolutionary history, systematics andbiogeography. We tested the impact of Gulf Group versus Mexican Group geography on rates of cladogenesis using amaximum likelihood framework, testing different models of birth/extinction of lineages. We propose a comprehensivephylogenetic hypothesis for the subfamily based on mitochondrial and nuclear loci (3,833 bp) using Bayesian and MaximumLikelihood methods. The phylogenetic structure found two phylogenetic groups associated to the two main geographiccomponents (Gulf Group and Mexican Group) and is partially consistent with the historical structure of river basins. Theprevious hypothesis, which divided the genus into three subgenera based on genitalia morphology was only partiallysupported (P = 0.047), resulting in a paraphyletic subgenus Pandicambarus. We found at least two cases in whichphylogenetic structure failed to recover monophyly of recognized species while detecting several cases of cryptic diversity,corresponding to lineages not assigned to any described species. Cladogenetic patterns in the entire subfamily are betterexplained by an allopatric model of speciation. Diversification analyses showed similar cladogenesis patterns between bothgroups and did not significantly differ from the constant rate models. While cladogenesis in the Gulf Group is coincident intime with changes in the sea levels, in the Mexican Group, cladogenesis is congruent with the formation of the Trans-Mexican Volcanic Belt. Our results show how similar allopatric divergence in freshwater organisms can be promotedthrough diverse vicariant factors.

Citation: Pedraza-Lara C, Doadrio I, Breinholt JW, Crandall KA (2012) Phylogeny and Evolutionary Patterns in the Dwarf Crayfish Subfamily (Decapoda:Cambarellinae). PLoS ONE 7(11): e48233. doi:10.1371/journal.pone.0048233

Editor: Jose Castresana, Institute of Evolutionary Biology (CSIC-UPF), Spain

Received December 22, 2011; Accepted September 27, 2012; Published November 14, 2012

Copyright: � 2012 Pedraza-Lara et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: Carlos Pedraza-Lara was partially supported by a predoctoral grant provided by CSIC, Spain. The funders had no role in study design, data collectionand analysis, decision to publish, or preparation of the manuscript. No additional external funding received for this study.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: [email protected]

Introduction

The freshwater crayfish subfamily Cambarellinae is comprised

of the unique genus Cambarellus, with 17 recognized species and a

disjunctive distribution across the freshwater streams of the Gulf

Cost of the United States and North and Central Mexico (Fig. 1)

[1]. The subfamily is unique because of the exceptionally small

body size of its species. They typically reach only 4 cm compared

to most crayfish averaging a maximum body size of .5 cm; hence,

the reference to the genus as the ‘‘Dwarf’’ crayfishes. Their

distribution goes from the Swanee River in northern Florida,

eastward through the southern Mississippi River watershed to

southern Illinois and continues southwest to the Nueces River in

Texas [2,3]. In Mexico, Cambarellus has a discontinuous distribu-

tion with three distant and isolated populations from the northern

states of Chihuahua, Coahuila and Nuevo Leon and then along

the Trans-Mexican Volcanic Belt (TMVB) [4,5]. The genus

contains species largely inhabiting lakes and lentic habitats. The

evolutionary history of such a broad and disjunct distribution of

species is unclear and our goal with this study is to shed some light

on the biogeography of the Cambarellinae.

A series of apomorphic morphological characters define the

subfamily and, therefore, monophyly has been accepted since its

proposal. These include, as for other crayfish groups, genital

morphology, which is particularly important, but also a small body

size, specific branchial formula, movable and enlarged annulus

ventralis (female genitalia) and the absence of the cephalic process in

the first pair of pleopods (male genitalia) [1,2,6]. The morpholog-

ical unity of these characters that define the subfamily contrasts

with the wide morphological variation in other characters

described for populations of several species [2,5,7]. This diversity

within and among species makes designation and identification

difficult, especially for widely distributed species [2,5].

Despite the intriguing geographic distribution and species

diversity in the Cambarellinae, the only phylogenetic hypothesis

for species relationships in the group is based on phenotypic

information and genital morphology [2]. With this hypothesis

(Fig. 2) three subgenera were proposed; Pandicambarus (containing

seven species), the monotypic Dirigicambarus, both comprised of

species occurring north of the Rio Grande (the Gulf Group), and

Cambarellus, containing species south of the Rio Grande (the

Mexican Group) [2]. However, no apomorphic characters have

PLOS ONE | www.plosone.org 1 November 2012 | Volume 7 | Issue 11 | e48233

Figure 1. Map of localities sampled. Map of localities sampled in this study, numbers are referred to in Table 1. Sample locations are colored torepresent different clades recovered by phylogenetic analyses (see Fig. 3). Open circles correspond to the only locality records for the three speciesnot included in the analyses as they were not found during sampling, or did not amplify during PCR reactions. Gray background refers to elevation(500–6000 m).doi:10.1371/journal.pone.0048233.g001

Evolutionary Patterns in Cambarellinae


Table 1. Sampling localities and Genbank accession numbers from individuals of Cambarellus used in this study.

+Species id fromthis study

Subgenus(Fitzpatrick,1983) GeneBank accession numbers

16S 12S cox1 28S H3

1 Cambarellus blacki Pandicambarus JX127836 JX127697 JX127977 JX127568 JX127429

1 Cambarellus blacki Pandicambarus JX127837 JX127698 JX127978 JX127569 JX127430

2 Cambarellus diminutus* Pandicambarus JX127810 JX127953 JX127545 JX127405

3 Cambarellus lesliei* Pandicambarus JX127809 JX127952 JX127544 JX127404

4 Cambarellus ninae Pandicambarus JX127814 JX127957 JX127549 JX127409

5 Cambarellus ninae** Pandicambarus JX127833 JX127694 JX127974 JX127565 JX127426

6 Cambarellus puer1233 Pandicambarus JX127822 JX127686 JX127965 JX127557 JX127417

7 Cambarellus puer Pandicambarus JX127815 JX127958 JX127550 JX127410

8 Cambarellus schmitti Pandicambarus JX127811 JX127954 JX127546 JX127406

9 Cambarellus schmitti Pandicambarus JX127838- JX127699- JX127979- JX127570- JX127431-

JX127855 JX127714 JX127996 JX127587 JX127447

10 Cambarellus schmitti Pandicambarus JX127856 JX127715 JX127997 JX127448

11 Cambarellus texanus Pandicambarus JX127832

12 Cambarellus texanus*** Pandicambarus JX127834 JX127695 JX127975 JX127566 JX127427

13 Cambarellus texanus Pandicambarus JX127819 - JX127683 – JX127962 – JX127554 – JX127414 –

JX127821 JX127685 JX127964 JX127556 JX127416

14 Cambarellus shufeldtii Dirigicambarus JX127812 JX127955 JX127547 JX127407



17 Cambarellus shufeldtii Dirigicambarus

18 Cambarellus shufeldtii Dirigicambarus JX127835 JX127696 JX127976 JX127567 JX127428



21 Cambarellus zempoalensis Cambarellus JX127725 JX127599 JX127868 JX127460 JX127320









25 Cambarellus zempoalensis Cambarellus JX127744 JX127618 JX127887 JX127479, JX127339








28 Cambarellus zempoalensis Cambarellus JX127771 JX127645, JX127914 JX127506 JX127366









Table 1. Cont.



16S 12S cox1 28S H3


31 Cambarellus patzcuarensis Cambarellus JX127728 JX127602 JX127871 JX127463 JX127323









35 Cambarellus sp. (cladeIII) Cambarellus JX127738 JX127612 JX127881 JX127473 JX127333

35 Cambarellus sp. (cladeIII) Cambarellus JX127752 JX127626 JX127895 JX127487 JX127347

36 Cambarellus chapalanus Cambarellus JX127726 JX127600 JX127869 JX127461 JX127321



















43 Cambarellus prolixus Cambarellus JX127729 JX127603 JX127872 JX127464 JX127324














51 Cambarellus sp. (clade V) Cambarellus JX127780 JX127654 JX127923 JX127515 JX127375




been proposed to support these subgeneric classifications and no

formal phylogenetic hypothesis has been evaluated using either

molecular or morphological characters. Therefore, we propose to

estimate a robust phylogenetic hypothesis for the group using an

extensive molecular data set. We then use this phylogenetic

framework to evaluate a coherent taxonomy for the group and to

test biogeographic hypotheses regarding the origin and spread of

the dwarf crayfish.

We also examine diversification patterns in the subfamily

through the estimated phylogenetic history of the species within

the subfamily. Phylogenetic diversity patterns are impacted by

geographic features and geologic history due to their effects on

allopatric speciation [8]. Given the contrasting geographical

features (Fig. 1) coupled with their distinct geological histories

occupied by the different groups in Cambarellinae, we will use

reconstructed molecular phylogenies to serve as models of lineages

through time (LTT), that will allow us to test the tempo and

Table 1. Cont.



16S 12S cox1 28S H3

53 Cambarellus sp. (clade V) Cambarellus JX127787 JX127661 JX127930 JX127522, JX127382


54 Cambarellus sp. (clade VI) Cambarellus JX127730 JX127604 JX127873 JX127465 JX127325



55 Cambarellus montezumae Cambarellus JX127731 JX127605 JX127874 JX127466 JX127326





57 Cambarellus sp. (clade VIII) Cambarellus JX127727 JX127601 JX127870 JX127462 JX127322

57 Cambarellus sp. (clade VIII) Cambarellus JX127767 JX127641 JX127910 JX127502 JX127362

58 Cambarellus occidentalis Cambarellus JX127784 JX127658 JX127927 JX127519 JX127379

58 Cambarellus occidentalis Cambarellus JX127785 JX127659 JX127928 JX127520 JX127380

59 Cambarellus occidentalis Cambarellus JX127813 JX127956 JX127548 JX127408

Procambarus toltecae JX127823 JX127687 JX127966 JX127558 JX127418

Procambarus acutus1 JX127824 JX127688 JX127967 JX127559 JX127419

Procambarus acutus2 JX127827 JX127970 JX127562 JX127422

Procambarus llamasi1 JX127825 JX127689 JX127968 JX127560 JX127420

Procambarus llamasi2 JX127826 JX127690 JX127969 JX127561 JX127421

Procambarus clarkii JX127829 JX127692 JX127971 JX127563 JX127424

Procambarus bouvieri JX127828 JX127691 JX127423

Orconectes deanae JX127859 JX127717 JX128000 JX127590 JX127451

Orconectes ronaldi JX127865 JX127722 JX128005 JX127596 JX127457

Orconectes virilis1 JX127866 JX127723 JX128006 JX127597 JX127458

Orconectes virilis2 JX127860 JX127591 JX127452

Cambarus brachydactylus++ DQ411732 DQ411729 DQ411783 DQ411802

Cambarus maculatus JX127864 JX127721 JX128004 JX127595 JX127456

Cambarus pyronotus JX127862 JX127719 JX128002 JX127593 JX127454

Cambarus striatus JX127861 JX127718 JX128001 JX127592 JX127453

Fallicambarus byersi JX127863 JX127720 JX128003 JX127594 JX127455

Fallicambarus caesius JX127867 JX127724 JX128007 JX127598 JX127459

Fallicambarus fodiens JX127858 JX127716 JX127999 JX127589 JX127450

+Locality number, as depicted in Figure 1.*Type specimens or type localities.**Morphologically identified as C. shufeldtii.***Morphologically identified as C. puer.++Sequence from the study of Buhay et al. 2007, tissue originally from the Carnegie Museum of Natural History.Populations termed as ‘C. sp’ are new proposed taxa, according to phylogenetic structure (see Figure 3).Populations from clade I are included in the lineage of C. zempoalensis, species which has to be re-examined by incorporing C. montezumae lermensis in the analysis.doi:10.1371/journal.pone.0048233.t001



pattern of change across lineages [9,10,11]. In the present study,

we used our molecular dataset on the subfamily Cambarellinae to

infer the timing and mode of lineage accumulation (patterns of

speciation minus extinction) which allows us to determine whether

there have been contrasting patterns in rates of diversification

between the two geographical components of this group; namely,

those defined as the Gulf and Mexican Groups, as a result of

contrasting biogeographic histories. Finally, we identify a geolog-

ical timescale consistent with biogeographic factors and cladoge-

netic events in this group.

Materials and Methods

Sampling and SequencingNo specific permits were required for the described field studies,

as none of the studied species were included in any endangered

list, at national or international levels at the time of sampling

(comprising the years 2005 and 2006). Including field and museum

localities, 59 geographic locations covering 14 of the 17 species

were collected throughout the distributional range of the subfamily

Cambarellinae (Fig. 1). Taxonomic identification was carried out

using existing keys [12]. The two main ranges for the subfamily

were covered, along the Neartic and the Transition zone of North

America, from the Mississippi River basin to the TMVB in central

Mexico. Most of the species could be sampled, but those tissues

from species with very restricted distribution ranges and/or being

collected in a reduced number of times in wild were obtained from

museum specimens (National Museum of Natural History,

Smithsonian Institution) (Table 1). Detailed data about samples

included are summarized in the Table S1.

The central goal of this work is to estimate a robust phylogenetic

hypothesis for relationships among the species within the subfamily

to test taxonomic hypotheses, biogeographic hypotheses, and

speciation hypotheses. As phylogenies are most accurately

estimated using broad taxonomic sampling as well as extensive

character sampling, we attempted to sample all species within the

subfamily (but are missing three of them) and collected sequence

data from five different gene regions (three mitochondrial and two

nuclear). We sequenced the mitochondrial genes 16S rDNA (16S),

12S rDNA (12S) and Cytochrome Oxidase subunit I (COI). These

genes have good phylogenetic signal in crustaceans [13] and are

considered optimal choices to characterize the genetic variation in

crustacean groups. Nuclear genes sequenced were 28S rDNA large

ribosomal unit (28S) and Histone 3 (H3) gene, which also have

some variation among species and are particularly good at

discerning deeper nodes [13].

PCR amplifications using gene specific primers (Table 2) were

carried out in 25 mL reactions containing: 16PCR buffer, 0.5 mM

of each primer, 0.2 mM of each dNTP, 1.5 mM MgCl2, 1 U Taq

Figure 2. Morphologic hypothesis tested. Phylogenetic hypothesis based on morphologic analysis of the monotypic subfamily Cambarellinae(genus Cambarellus), indicated are the subgenera previously proposed, mainly based on genital morphology (Fitzpatrick, 1983).doi:10.1371/journal.pone.0048233.g002



polymerase (Biotools), and about 10–50 ng of template DNA. The

cycling profile for PCR amplifications was 3 min at 94uC (1 cycle),

30 s at 94uC, 30 s at the primer-specific melting temperature and

60 s at 72uC (30 cycles), followed by a final extension of 4 min at

72uC. PCR products were visualized in 1.0% agarose gels

(16TBE) and stained with SYBR-Safe (Invitrogen). Fragments

were sequenced on an ABI 3730XL DNA Analyzer. Sequences of

the different gene fragments were aligned using MUSCLE [14]. In

the case of the COI gene, recommendations to detect the

occurrence of possible nmtDNA were carried out for each

sequence. These included the identification of stop codons,

repeated sequencing of samples, nonsynonymous substitution

and unusual levels of genetic divergence in samples from the

same population [15,16].

Phylogenetic AnalysesPartition homogeneity tests were carried out on the concate-

nated matrix using PAUP v. 4.0b10 [17]. We examined

homogeneity across partitions by gene and by codon position for

protein-translated fragments (Table 3). We estimated phylogenies

using Maximum Likelihood (ML) and Bayesian Inference (BI)

approaches. Additionally, we used 15 species of the family

Cambaridae as outgroups: Cambarus maculatus, C. striatus, C. pyr-

onotus, C. brachidactylus, Orconectes ronaldi, O. virilis, O. deanae,

Fallicambarus caesius, F. fodiens, F. byersi, Procambarus bouvieri, P.

clarkii, P. llamasi, P. acutus and P. toltecae (Table 1).

In order to identify the most appropriate evolutionary model of

nucleotide substitution (Table 2), we considered the Akaike

corrected information criterion (AICc) [18], and the Bayesian

Information Criterion (BIC) [19] as estimated using the program

jModeltest [20]. A phylogenetic tree was constructed under ML

using PHYML 3.0 [21] and AICc-selected parameters for the

concatenated matrix. The tree search was started with an initial

BIONJ tree estimation followed by a Subtree Pruning and

Regrafting (SPR) topological moves algorithm. We assessed

confidence in branches using 1000 nonparametric bootstrap [22]

replicates under the best-fit evolutionary model.

Bayesian inference of phylogeny was implemented in MrBayes

v. 3.1.2 [23], following the BIC-selected parameters and applying

a Monte Carlo Markov Chain (MCMC) search procedure for 10

million generations. Sequences were partitioned by codon position

for COI and by gene for the rest of fragments, using the

parameters found by BIC as priors and unlinking the run

parameters. Convergence between the different run parameters

in paired simultaneous runs (4 chains by run), trees were sampled

every 100 generations and run length was adjusted considering an

adequate sampling based on average standard deviation of split

frequencies being ,0.01 [24]. We examined the results and

determined the burn-in period as the set of trees saved prior to log

likelihood stabilization and convergence as estimated using Tracer

1.4.1 [25], eventually the first 10% trees. Tracer was also used to

check for convergence between chain runs and optimal values of

run parameters. Confidence in nodes was assessed from the

posterior probabilities along the MCMC run. Highly supported

nodes are termed herein as those with a value of 95% or more in

posterior probabilities and bootstrap values.

We tested our resulting topology against the phylogenetic

hypotheses put forth by Fitzpatrick [2]; namely, the three

subgenera are monophyletic and show the following relationships

((Dirigicambarus, Pandicambarus),Cambarellus). Topology constrained

ML scores were estimated for each hypothesis in PAUP*.

Congruence with alternative hypotheses was evaluated in a ML

framework applying the Shimodaira-Hasegawa (SH; [26]) test and

the Approximate Unbiased (AU) test [27] with 50,000 RELL

bootstrap replicates as implemented in TreeFinder [28]. We also

tested these hypotheses using a Bayesian approach by identifying

the alternative hypothesis within the set of Bayesian tree topologies

and testing for significant differences. To do so, we filtered the

post-burnin Bayesian topologies included in the set of trees with

the constraint topology in PAUP* [17].

Divergence DatingIn order to propose an accurate time frame for phylogenetic

divergence processes, we estimated mean node ages and their 95%

highest posterior densities (HPDs) using Bayesian relaxed molec-

ular clock methods [29] as implemented in BEAST ver. 1.6.1 [30].

In this method, tests of evolutionary hypotheses are not

conditioned on a single tree topology, which allows for simulta-

neous evaluation of topology and divergence times while

incorporating uncertainty in both. A uniform Yule tree prior

was specified, as appropriate for hierarchical rather than reticulate

relationships, and a subsampling of one representative of every

lineage was included to avoid over-representation of certain

individual lineages with more sampling. We applied the optimal

model of data partitioning and DNA substitution identified by BIC

for each gene (COI, 16S, 12S, 28S and H3) and for codon

positions for COI. An uncorrelated relaxed lognormal molecular

clock was applied to model rate variation across branches, and

pertinence of a relaxed estimation was checked after verifying that

the distribution of the coefficient of variation was .1. The dating

analysis was performed with the total matrix, but calibration of the

molecular clock was done using COI and 16S mutation rates only,

as information on rates of mutation of these two fragments is

widely described in multiple groups and for which there is

extensive fossil calibrated divergence time data in crustaceans

Table 2. Primer and PCR conditions used in this study toamplify different gene regions.

Generegion primers sequence Tm(6C) Reference

COI COIAR GTTGTTATAAAATTHACTGARCCT 48.5 This study

COIBF GCYTCTGCKATTGCYCATGCAGG 48.5 This study

COIBR TGCRTAAATTATACCYAAAGTACC 48.5 This study

COICF ACCTGCATTTGGRATAGTATCTC 48.5 This study

COICR GAAWYTTYAATCACTTCTGATTTA 48.5 This study

COIDF CTGGRATTGTTCATTGATTTCCT 48.5 This study

ORCO1F AACGCAACGATGATTTTTTTCTAC 48.5 [75]

ORCO1R GGAATYTCAGMGTAAGTRTG 48.5 [75]

16S 1471 CCTGTTTANCAAAAACAT 46 [76]

16S-1472 AGATAGAAACCAACCTGG 46 [76]

12S 12sf GAAACCAGGATTAGATACCC 53 [77]

12sr TTTCCCGCGAGCGACGGGCG 53 [77]

28S 28s-rD1a CCCSCGTAATTTAAGCATATTA 52 [78,79]

28s-rD3b CCYTGAACGGTTTCACGTACT 52 [78,79]

28s-rD3a AGTACGTGAAACCGTTCAGG 52 [78,79]

28s-rD4b CCTTGGTCCGTGTTTCAAGAC 52 [78,79]

28sA GACCCGTCTTGAAGCACG 52 [78,79]

28S B TCGGAAGGAACCAGCTAC 52 [78,79]

H3 H3 AF ATGGCTCGTACCAAGCAGACVGC 57 [80]

H3 AR ATATCCTTRGGCATRATRGTGAC 57 [80]

doi:10.1371/journal.pone.0048233.t002



[31,32]. As a representation of these substitution rates, we

considered the range to include extreme values reported, which

extends between 0.23–1.1% per million years (PMY) for 16S

[33,34] and 0.7–1.3% PMY for COI [35,36,37]. These sets were

introduced as uniform prior distributions, as no evidence justifies a

specific distribution of rates in our data, avoiding the introduction

any additional bias to the rate values assumed. Considering the

geographic distribution of the genus, a geological calibration was

also included as identified with the uplifting of the TMVB, which

began around 12 MYA [38]. This age was set as a maximum for

MRCA of the Mexican species. Additionally, fossil calibration was

included in one point as the minimum age to account from the

oldest fossil from the genus Procambarus [a Procambarus primaevus,

52.6–53.4 MYA, [39]]. Monophyly was not enforced for any

node. Analyses were run for 20 million generations with a

sampling frequency of 2000 generations. Tracer was used to

determine the appropriate burn-in by monitoring run parameters

by ensuring all effective sample sizes (ESS) were larger than 200

and independent runs converged. Two million generations were

discarded before recording parameters and four independent runs

were performed to ensure values were converging on similar

estimates.

Diversification PatternsThe two main components of the subfamily occupy two regions

highly contrasting in topography and biogeographic history. Thus,

a second objective in this study was to describe the patterns of

cladogenesis involved in the evolutionary history of Cambarellinae

and to test the hypothesis that the different biogeographic histories

from the two different geographic ranges of the subfamily (i.e., the

Mexican and Gulf Groups), could lead to contrasting cladogenetic

patterns evidenced by possible diversification shifts. Shifts in birth

and death rates can leave distinctive signatures in phylogenies,

resulting in departures from linearity in semi-log LTT plots [9,11].

We compared diversification rates from the reconstructed

phylogeny of the entire subfamily and of the two main clades

(Mexican Group vs. Gulf Group) to different null models of

diversification by using the Birth-Death Likelihood method (BDL).

This temporal method was used to test different hypothesis of

cladogenesis rate shifts [40]. BDL uses maximum likelihood

estimates of speciation rate parameters and a likelihood score per

tree, and test different rate-variable models against null models of

rate-constancy under the Akaike Information Criterion (AIC) [18].

To provide an indication of the diversification rates in each case,

we generated a logarithm LTT plot using the LASER package

version 2.2 [41]. The LTT plot was generated from the Maximum

Clade Credibility tree from BEAST, after pruning the terminals

not included in each clade tested using TreeEdit v1.0a10 [42] and

rooting the basal age to the one observed from the dating analysis.

To test for significant departures from the null hypothesis of rate-

constancy, observed DAICRC from our data was compared to

those from the different rate diversification models using BDL as

implemented in the LASER package version 2.2 [41]. The test

statistic for diversification rate-constancy is calculated as:

DAICRC =DAICRC2DAICRV, where AICRC is the Akaike

Information Criterion score for the best fitting constant-rate

diversification model, and AICRv is the AIC for the best fitting

variable-rate diversification model. Thus, a positive value for

DAICRC indicates that a rate-variable model best approximates

the data. We tested five different models, of which two are rate-

constant and three are rate-variable: 1) the constant-rate birth

model (Yule) [the Yule process; [43]] with one parameter l and mset to zero; 2) the constant-rate birth-death model with two

parameters l and m (BD); 3) a pure birth rate-variable model

(yule2rate) where the speciation rate l1 shifts to rate l2 at time ts,

with three parameters (l1, l2, ts); density-dependent speciation

models with two variants, 4) exponential (DDX) and 5) logistic

(DDL). Significance of the change in AIC scores was tested by

generating a distribution of scores. This was done through

simulation of 9000 trees using yuleSim in LASER, for the entire

Cambarellinae subfamily and each geographic group, reflecting

our sampling size in each case and having the same speciation rate

as under the pure-Birth model.

Results

PhylogenyWe sequenced three mitochondrial (16S (519 bps), 12S

(365 bps) and COI (1527 bps)) and two nuclear (28S 1100 bps

and H3 322 bps) gene fragments resulting in 3833 characters

(2411 mitochondrial and 1422 nuclear) and giving a series of

substitution models (Table 3). These new data have been deposited

in GenBank (Table 1). COI-like sequences were found in seven

cases, identified by the occurrence of one or several stop-codons

along the sequence and an unusual sequence divergence, which

affected position in the tree and divergence regarding the other

sequences coming from the same population. These sequences

were removed from data sets and not considered for any analysis.

As previously reported [15], when working with COI sequences in

crayfish these sequences have to be specially checked to ensure

they are mitochondrial.

The most variable fragment was 12S, followed by COI and 16S

(variable sites: COI = 530/1527, 16S = 199/519 12S = 143/365;

besides this, COI showed the highest proportion of parsimony

informative (PI) sites: COI = 419, 16S = 121, 12S = 80) (Table 3).

As expected, nuclear fragments were the most conservative (for the

Table 3. Substitution model and phylogenetic performance of each gene fragment.

Gene Size (pb) Substitution model/gamma parameter/Invariable sites Variable sites PI %PI

AICc BIC

16S 501 HKY+G; 0.232 HKY+G; 0.230 199 121 24.1

12S 358 K80+G; 0.219 TVM+G; 0.213 143 80 22.3

COI 1527 HKY+G; 0.321 HKY+G; 0.321 530 502 32.8

28S 992 TIM3+G; 0.031 TIM3+G; 0.031 39 28 2.8

H3 322 JC; – HKY+I; 0.834 31 24 7.4

All 3700 GTR+G; 0.256 GTR+G; 0.254 1431 847 22.8

doi:10.1371/journal.pone.0048233.t003



mitochondrial set, variable sites = 1187, PI = 783; for the nuclear

set, variable sites = 244, PI = 64). The complete combined data set

contained 1431 variable sites (,37%), and 847 PI (,22%).

The topologies recovered by mitochondrial and nuclear

analyses based on ML and BI methods were similar (Figure 3),

although some discrepancies can be found in some terminal taxa

arrangements and between genera-outgroup relationships, princi-

pally concerning the relative positions of Cambaridae genera

representatives. Both topologies show Cambarellus as a monophy-

letic clade (Figure 3). Within Cambarellus we found two divergent

clades which correspond to the two distinct geographic ranges of

the genus based on a highly supported node by ML and BI

analyses (more than 95% of nodal support values). The first

lineage included the species from the Mexican Group, coincident

with the TMVB in Mexico. The second lineage included the Gulf

Group, containing the species distributed in USA. Only results

from the combined analyses of mitochondrial and nuclear

information are shown, as nuclear evidence did not have enough

phylogenetic signal to distinguish relationships within each

geographic group (Mexican and Gulf Groups). As shown in

different studies, mitochondrial and nuclear information could

resolve different portions of the phylogeny (i.e., shallow vs. deep

levels of tree, [44,45] ) and that was one of the major reasons for

combining these data types in this study. The hypothesis

explaining this is that long-branch attraction might be more

common among deeper nodes, and that slow-evolving nuclear

DNA might help to resolve such issues [46,47].

Topology tests rejected the null hypothesis of an equally good

explanation for all the constrained and the unconstrained

topologies. The topology obtained in this study showed a

significantly better Likelihood score (L = 227483.1) than the

monophyletic grouping of Pandicambarus subgenus. Our phyloge-

netic estimate resulted in a monophyletic subgenus Cambarellus and

Dirigicambarus, but Dirigicambarus was nested within the paraphyletic

Pandicambarus (Fig. 3). We tested the monophyly of the Pandicam-

barus by forcing this alternative topology and we can reject this

hypothesis by the results of SH and AU tests (likelihood values for

the alternative hypothesis/p values for SH and AU - 27565.1/

0.043, 0.047). Except for the division within Pandicambarus,

Fitzpatrick’s notion of relationships among the subgenera is

supported by our resulting topology, except for the non-

monophyletic Pandicambarus as Pandicambarus and Dirigicambarus

are nested together as a sister clade that is then sister to Cambarellus

as proposed by Fitzpatrick. Bayesian inference also failed to

support the monophyly of Pandicambarus failing to find a

monophyletic Pandicambarus in 9900 trees resulting from the

MCMC search.

Species were generally well recovered as monophyletic groups

for most of those included in the Gulf Group, but a different

situation is depicted for the Mexican Group (Figure 3). The clades

highly supported by phylogenetic analyses have a geographic

concordance, supporting the hypothesis that geographic events

could have been important factors influencing cladogenesis in the

genus, especially those regarding geographic features of the

TMVB. Phylogenetic structuring between all Mexican taxa did

not support the monophyly of some of the species currently

recognized, as the highly supported clades showed representatives

of multiple named species, suggesting that some of the named

species did not form monophyletic assemblages.

Low 16S divergences can be observed between taxa. Diver-

gences obtained between those contained in the Gulf Group were

higher than those from the Mexican Group. The mean sequence

divergence considering the likelihood model within the former was

DHKY = 4.13%, and that within the latter was DHKY = 1.18%

(Table 4).

The Mexican Group is composed of several clades highly

supported by ML and BI analyses (95–100% support, termed with

roman numerals in Figure 1), which also show geographic

concordance. Some geographic overlapping between clades was

observed, mainly along the Lerma Basin. The Clade I included

populations from the Cuitzeo and Middle-Lerma basins, morpho-

logically assigned to C. montezumae. C. zempoalensis from type locale

was placed inside this clade as well. Cambarellus patzcuarensis from

the basins of Patzcuaro and Zirahuen were contained in Clade II

and sister clade to Clade I. The third and more divergent clade

(Clade III) consisted of a population from La Mintzita, geograph-

ically close to the Cuitzeo basin.

Clade IV consisted of populations from the basin of Chapala

and its tributaries (Duero River), as well as its neighboring basins,

Cotija and Zapotlan. This group included two species, C.

chapalanus and C. prolixus, both found in Lake Chapala and

associated with different habitat conditions. Also included here

were populations from up-stream tributaries of the Santiago River,

which originates as an outflow of the Chapala Lake. Clade V

contained populations from the river Ameca basin. Clade VI

contained the population from Zacapu Lagoon. The Clade VII

included two populations from the eastern-limits of the distribution

of the genus in the TMVB, the populations of Xochimilco (type

locality for C. montezumae) from the Valley of Mexico basin and the

crater lake Quechulac. The Clade VIII was composed of two

populations from the northern margin of the Middle-Lerma basin

and the Clade IX by populations from the basins of the Santiago

and Magdalena rivers, in the west part of TMVB.

Gulf Group relationships depict a phylogenetic structuring

corresponding to geographic ranges. C. diminutus corresponds to

the most divergent lineage, while two clades were recovered with

high ML and BI support corresponding to a west-east pattern. The

first clade contained most of the species from the Central and East

Gulf Coast (CEG), except C. diminutus, and included four

recognized species. Populations of C. shufeldtii from the Mississippi

river basin form a monophyletic group, while C. blacki, C. lesliei,

and C. schmitti are grouped together in a sister clade to the latter,

geographically covering the eastern extreme distribution range of

the genus in the Gulf Group from the Mobile Bay, Alabama to the

Swuanee River, Florida. A similar grouping is observed in the

second clade of the Gulf Group, containing populations from the

West Gulf Coast (WG), mainly in the south-west part of Texas,

where C. puer was recovered as a sister lineage to the clade

grouping C. texanus and C. ninae.

Diversification Patterns and DatingLog-likelihood scores with the molecular clock enforced and not

enforced were 213.893 and 213.767, respectively. As the LRT

rejected the null hypothesis of a global molecular clock (x2 252,

P = 0.001), the sequences analyzed did not evolve at a homoge-

nous rate along all branches and we proceeded to use a relaxed

molecular clock (Fig. 4) as a result.

Ages from the dating analysis were recovered with consistency

through repetitions (Figure 4). The crown age for the tree was

53 Myr (95% highest posterior density [HPD] interval for node

heights/ages: 52.6–53.7 Myr), which corresponds to the separa-

tion of the genus Procambarus from the rest of the groups. We

estimated an approximate age of 31.0 Myr (27.4–34.9 Myr 95%

HPD) for the TMRCA of clade containing the Cambarellinae.

MRCA for the terminals included in the two lineages of the Gulf

Group is approximately 16.7 Myr (13.9–19.7 Myr 95% HPD).

MRCA of the Mexican Group was dated around 11.1 (9.8–





11.9 Myr 95% HPD). We propose some major biogeographic

events inferred from the phylogenetic structure, which depicted

different vicariant and dispersion events along the evolutionary

history of Cambarellinae (depicted in Figure 4.).

The LTT plots track the temporal accumulation of lineages in a

clade and indicate that the subfamily Cambarellinae did not

significantly deviate from a constant model of diversification

during its evolutionary history, as evidenced in the LTT analyses

for the entire subfamily (including both, Gulf and Mexican

Groups, see Fig. 5). LTTs rate-constancy models received better

AIC scores, and they were not significantly different from the best

rate-variable model for all analyses (Table 5). The pure birth

speciation rate model was identified as having the lowest AIC

value amongst the other models tested for the subfamily together

and the two groups separately. Although the Mexican Group

showed the highest diversification rate (under pureBirth model

r = 0.174), it is still a low value as compared to recognized shifts in

diversification in other animal groups ranging from 0.4 to 0.8

speciation events per million years [48,49].

Quick inspection of the LTT plots shows some differences

between the cladogenesis of the entire subfamily and that of the

Gulf and Mexican Groups alone (Figure 5). However, according to

the BDL analysis, the diversification rate-constancy statistic

DAICRc was found to be similar between them, being 20.135

for the entire subfamily, 21.38 for the Mexican Group and 21.36

for the Gulf Group, indicating that the data are a better fit to the

constant rather than variable rate model of diversification in all

cases. Goodness-of-fit tests indicated that the mean Bayes LTT

from the entire subfamily was not significantly different from

expectations under any of the rate constancy models (AIC

pureBirth and BD = 35.20 and 35.63, respectively). The values

from the BDL analysis of the Mexican and the Gulf Groups were

not significantly different than the critical values found under the

different simulated constant rate models (for AIC pure-

Birth = 22.40 and BD = 24.40 for the Mexican Group and AIC

pureBirth = 26.20 and BD = 28.17 for the Gulf Group). These

results are consistent with a lack of evidence about episodes of

shifts in diversification rates along the evolutionary history of

Cambarellinae or its two groups separately.

Discussion

Phylogenetic RelationshipsOur results are consistent with the monophyly of the

Cambarellinae subfamily, previously proposed from morphology

and a set of apomorphic characters [2]. The combination of

mitochondrial and nuclear markers provide sufficient information

to resolve the relationships between highly supported clades,

namely the Gulf (Pandicambarus/Dirigicambarus) and Mexican

(Cambarellus) Groups and included clades (Figure 3). Less resolution

is observed at the deeper nodes of the Mexican Group, where

several clades were not supported by all analyses. It is possible, as

commonly argued for polytomies, that such patterns could be

related to an acceleration of speciation rates in a short period of

time [50]. Species sampling in this study is not complete, as three

species are still to be added to the phylogenetic analysis. These

correspond to C. alvarezi, C. areolatus and C. chihuahuae from North

of Mexico and have almost no collection records. Populations

from the aforementioned species are currently under serious threat

or possibly extinct, as we did not find any specimens in our

attempts to collect them. Their rarity is possibly due to extreme

habitat alteration or drought, a situation reported as critical for

freshwater fauna in some of the localities from where they have

been recorded [51,52]. Their future inclusion, if possible (mostly

through museum collections or captive populations), could provide

valuable insight into the phylogenetic relationships within the

subfamily, especially between the Mexican and Gulf Groups

defined here.

Several differences can be found between the phylogenetic

relationships emerging from this work and the previous hypothesis

[2]. First, relationships between species in the Gulf Group are not

congruent with several assumptions made from morphology,

especially regarding the phylogenetic meaning of genitalia

variation. Although species are generally well recovered as

monophyletic, their relationships are not congruent. As evidenced

by topology tests carried out in this study, sister relationships

proposed by genital morphology between the two subgenera from

the Gulf Group (Pandicambarus and Dirigicambarus) is not supported.

Instead, Dirigicambarus (composed by C. shufeldtii) is recovered as a

sister taxon of a clade containing C. lesliei and C. schmitti. This

would leave the subgenus Pandicambarus as paraphyletic, ultimately

questioning also its phylogenetic validity. Maintaining of the

subgenus Dirigicambarus for C. shufeldtii could be also questioned, as

no phylogenetic evidence supports it, pointing out that genital

distinctiveness in this species could be the result of drift events or

selective processes along its history. Besides its proposition as a

member of a separate subgenus, C. shufeldtii has been recognized as

a derived rather than a plesiomorphic representative [2], an

assumption supported in this study. Therefore, we recommend

that the subgenus Dirigicambarus be disregarded and that the genus

Cambarellus should contain only two subgenera, namely Cambarellus

and Pandicambarus that correspond to the Mexican and Gulf clades,

respectively (resulting in Cambarellus shufeldtii being considered a

member of the subgenus Pandicambarus). Our phylogenetic results

support the hypothesis of C. diminutus as having plesiomorphic

character states for the Gulf Group. Its unique morphological

traits (outlined in [2]) are in agreement with this hypothesis.

Taxonomic ImplicationsNumerous species concepts have been proposed that emphasize

different features for delimiting species. Sometimes, this has led to

contrasting conclusions regarding species limits and the number of

species in many groups. A ‘unified species concept’ was advocated

that emphasizes the common element found in many species

concepts, which is that species are separately evolving lineages

[53]. This unified concept also allows the use of diverse lines of

evidence to test species boundaries [e.g., monophyly at one or

multiple DNA loci, morphological diagnosability, ecological

distinctiveness, etc. [53,54] and is the species concept we follow

in this study.

There were two cases in which the inferred topology did not

recover species’ monophyly in the Gulf Group. The first one

shown by one individual morphologically assigned to C. shufeldtii

(Locality 5, Colorado Basin), which grouped with individuals of C.

Figure 3. Phylogenetic tree of Cambarellus genus. Phylogenetic tree of Cambarellus based on three mitochondrial and two nuclear genes.Bootstrap support from ML (above) and Posterior Probabilities from Bayesian Inference (bellow) are indicated on each node. ***Stands for 95 or more,**for 85–94 and *for 75–84 support values from ML analyses. Drawings correspond to male genital morphology, which is the base for traditionaltaxonomy of subgenus and species in the group. Individual 5–1 was morphologically identified as C. shufeldtii, but is considered here as C. ninaebased on the phylogenetic position in tree.doi:10.1371/journal.pone.0048233.g003



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ninae and the other by one individual morphologically assigned to

C. puer (Locality 12, San Bernard Basin), grouped with individuals

of C. texanus. The most plausible explanation for this could be the

finding of introgression of C. shufeltii, supported by the overlapping

ranges of these species in east Texas. As a common consequence,

introgression between species with smaller ranges could be favored

when they share similar regions with widely distributed species like

C. shufeldtii and C. puer (e.g., [55]). The aforementioned hypothesis

needs to be supported with faster-evolving nuclear markers, which

allow the differentiation between species, and could be ap-

proached in the near future.

Figure 4. Molecular dating of cladogenetic events. Dates and major biogeographic events inferred during cladogenesis of the Cambarellinesubfamily. A) Ultrametric tree resulting from the dating analysis. Mean ages are indicated in each node (MYA), and 95% HDP intervals are shown asblue bars. Black dots indicate node used for calibration (oldest fossil recorded for Procambarus). Numbers correspond to localities and romannumerals to clades from phylogenetic tree (Figure 3). B) Major cladogenetic events inferred from phylogenetic structure and dating. Red names referto extinct lineages.doi:10.1371/journal.pone.0048233.g004



For the Mexican Group, the phylogenetic structure shows a

geographic correspondence. This observation supports the hy-

pothesis that cladogenesis in the group has been influenced by

geological history. This geographic correspondence could explain

why instead of recovering species, cladogenetic structure recovered

different hydrological units as monophyletic. This is the case for

the widely distributed C. montezumae, which is not recovered as

monophyletic, as several populations morphologically assigned to

this species were located in different clades in the Mexican Group.

In fact, several populations morphologically assigned to C. mon-

tezumae form a paraphyletic group, as C. zempoalensis is recovered

inside this group. Another example concerns C. prolixus, included

inside the wider genetic variation of C. chapalanus. However, the

striking morphological distinctiveness of C. prolixus suggests a very

recent processes of divergence between this species and C.

chapalanus which may be missed by the genetic markers used here

[see [56] for discussion on the relative importance of genetic

markers versus selected morphological differences in species

studies]. Based on an unified species criterion, we found support

for all described species in the Mexican Group from TMVB,

which match to the terminal clades in tree (Figure 3) plus

C. prolixus, which possesses contrasting morphological and ecolog-

ical features. These clades correspond to six described species: 1)

C. zempoalensis, corresponding to the population from Zempoala.

Temporarily, we consider this species as valid but this needs to be

confirmed with an analysis including the ‘lermensis form’ (in the

terms of Villalobos’ proposal) [57]. This is because when

considering the range of this clade, it probably includes the

aforementioned form, from the upper Lerma Basin. As such,

C. montezumae lermensis would be raised to species rank and C.

zempoalensis would stand as a junior synonym; all populations found

in clade I would temporarily correspond to C. zempoalensis, until

confirmation of the above mentioned issue regarding its synonymy

with C. montezumae lermensis; 2) C. patzcuarensis, for those populations

from the Patzcuaro basin; 3) C. chapalanus, from the basin of

Chapala and adjacent basins; 4) C. prolixus, from certain habitat

conditions at Chapala Lake; 5) C. montezumae, from the Valley of

Mexico and adjacent basins and 6) C. occidentalis, from the lower

part of the Rıo de Santiago basin, at the western extreme of the

distribution in Mexico. In addition, we found several monophy-

letic clades, and in congruence to the same criterion, we propose

they correspond to no recognized species, those from the terminal

clades in tree (Figure 3): 1) clade III, for the population from La

Mintzita spring; 2) clade V, for populations from Ameca basin; 3)

clade VI, for populations from the Zacapu Lagoon and 4) clade

VIII, for certain populations from the northern side of the Middle

Lerma Basin (populations of La Laja basin and Vegil, see Table

S1).

Figure 5. Diversification patterns through time. LTT plot for the Cambarellinae subfamily (green), the Mexican Group (yellow) and the GulfGroup (blue).doi:10.1371/journal.pone.0048233.g005



Rates of Cladogenesis and Contrasting CladogeneticForces

Unlike the cladogenetic structure, the rate at which cladogenesis

took place in Cambarellus does not seem to be affected by geologic

events. Even when most of the cladogenetic events in the Mexican

Group are probably the result of vicariance corresponding to

geological features as the formation of the TMVB, a geologic

region that has been proposed to affect cladogenesis in different

freshwater groups [58,59], this study has found no effect of

geologic events on speeding or reducing cladogenesis rates.

Although different in nature, and affected by contrasting

geographic ranges, vicariant events in both groups lead to similar

cladogenetic trajectories, demonstrating the impact of climatic and

geologic forces on allopatric speciation.

All these lines of geological evidence indicate that the historical

geographic range of the hypothesized ancestral species of

Cambarellinae in Mexico and the Southeast of the United States

have changed dramatically over time. Additionally, some other

effects could have played a roll in speciation in both groups.

Although the continental ice sheets during the Pleistocene glacial

periods in North America never extended into the study area,

these glaciations had some profound indirect effects in freshwater

faunas in Mexico and are hypothesized to have permitted dispersal

by stream captures, local inland or estuarine flooding, and

interconnecting drainages due to lowered sea levels during the

late Neogene [60].

BiogeographyOur results support that MRCA for the Cambarellinae existed

in the Eocene, ,40.4 MYA (35.2–45.7 MYA). A singular

biogeographic event inferred from this study comes from the

separation of the two major clades, which could be related to the

Eocene-Oligocene boundary, a transition documented to strongly

affect terrestrial, marine and freshwater dwellers, as evidenced by

significant extinctions and taxonomic turnovers in a wide range of

groups [61,62]. In this case, the formation of the Rio Grande Rift

could have vicariant effects on the ancestors of both groups.

We postulate that historical vicariant events are related to

change in geographical barriers and climate in both groups while

dispersal events of some species are responsible for occupying the

current wider range, with their current absence related to

extinction periods. While these biogeographic events are present

in both groups, contrasting vicariance and dispersal impacts on

distributions are not unusual for freshwater crayfishes [63]. Here

we explain some possible alternatives, inferred from congruence in

timing of cladogenetic events. Species cladogenesis in the Gulf and

Mexican Groups are best explained by an allopatric mechanism of

speciation because no overlap is observed between sister taxa in

Cambarellus [8]. Estimation of divergence times provides a temporal

scenario of these events, allowing for a relationship of earth history

with hypothesized vicariant mechanisms proposed to promote

allopatric speciation. We postulate that divergence patterns in

these groups are contrasting in several ways. First, date estimates

agree with a more ancient diversification in the Gulf Group than

in the Mexican Group, even when possible events related to

species diversification from Northern Mexico could not be

Table 5. Results of the Birth-Death Likelihood analysis based on fitting different diversification models to Cambarellinae and itscontaining groups (Gulf and Mexican Groups).

Group pureBirth BD DDL DDX yule2rate

Cambarellinae Parameters r1 = 0.122 r1 = 0.053 r1 = 0.122 r1 = 0.064 r1 = 0.043

a = 0.706 k = 476707.6 x = 20.297 r2 = 0.152

Ln(L) 16.600 15.815 16.601 15.969 14.668

AIC 235.201 235.631 237.201 235.939 235.336

DAIC 0 20.430 22 20.738 20.135

Gulf r1 = 0.106 r1 = 0.088 r1 = 0.197 r1 = 0.127 r1 = 0.153

a = 0.229 k = 12.126 x = 20.117 r2 = 0.059

st = 4.116

Ln(L) 12.101 12.089 11.783 12.083 11.395

AIC 226.202 228.179 227.567 228.166 228.791

DAIC 0 21.977 21.365 21.964 22.589

Mexican r1 = 0.174 r1 = 0.174 r1 = 0.276 r1 = 0.296 r1 = 0.210

a = 0.0 k = 21.007 x = 0.283 r2 = 0.120

st = 2.225

Ln(L) 10.201 10.201 9.894 10.049 9.834

AIC 222.402 224.402 223.789 224.096 225.669

DAIC 0 22 21.387 21.694 23.267

r = net diversification rate (speciation events per million years);a = extinction fraction;st = time of rate shift (MYA);k = carrying capacity prameter;x = rate change parameter;Ln(L) = Log-Likelihood;AIC = Akaike information criterion;DAIC = change in AIC relative to pureBirth.doi:10.1371/journal.pone.0048233.t005



inferred. It is possible that the latter predate the ones observed for

the Mexican Group. Second, while the Gulf Group cladogenesis

could be more related to climatic oscillations, orogenic impacts

could have been more important for diversification of the extant

species in the Mexican Group. The absence of Cambarellus from the

Rıo Grande basin could be explained by a generalized extinction

of its populations related to the high desiccation rate since the

Tertiary [64]. The ultimate evidence of that would be the presence

of C. chihuahuae from the Guzman basin in the Southern part of the

Rıo Grande Rift. This high extinction rate could explain the

current disjunct geographic pattern between the Mexican and

Gulf Groups. It seems reasonable to consider that as a

consequence of the extinction rate along the former contact zone

between the groups. It would not be surprising to find relict

populations from both groups if further sampling efforts in this

region could take place, which could modify their known range

and find regions containing both lineages.

Proposed Vicariant Events Promoting SpeciationThe first diversification event in the Gulf Group was dated to

Early Miocene, ,16.7 MYA (13.9–19.7 MYA), and corresponded

to the separation of the C. diminutus lineage. It is possible that

extinction events could explain the observation of a unique well

differentiated branch leading to C. diminutus, although a wider

genetic variation not yet sampled from this lineage could be

possible, which would be consistent with the wide morphological

variation previously observed [2]. Orogenic activity dating to this

period corresponds at the SE of United States with the formation

of the Edwards Plateau, and the Miocene increased activity along

the Balcones Fault. The next cladogenesis recorded for the Gulf

Group is congruent with Late Miocene times, approximately 8.7

MYA (7.3–10.3 MYA), originating the Western (WG) and Eastern

Gulf Coast (EG) species groups. These speciation events are

consistent with sea levels along the gulf coast driven by climatic

oscillations since the Middle Miocene. These were characterized

by a dramatic rise in sea level between 80 and 100 m above the

present day sea level [65,66]. As a consequence, a marine

incursion took place along the coast of the Gulf of Mexico, which

could be important in the split of West and Central-East

distribution ranges and keep them separated long enough to

induce a strong speciation event.

In the Late Miocene there was a sharp drop of 80–100 m below

present sea levels, extending Gulf of Mexico tributaries further

south. This southward extension of Gulf Coastal rivers created

connections between tributaries that were isolated during periods

of higher sea level. The Late Miocene drop in sea level correlates

with the estimated age of the first speciation events among the

extant species of Cambarellus from the Gulf Group. Later, the

Pliocene (2.5–5.5 MYA) was characterized by a 50–80 m rise

above current sea level, but this incursion lasted for only a short

time, approximately one million years [66]. Sea levels dropped in

the Late Pliocene, and during the Pleistocene there were at least

three major fluctuations in sea level, none rising higher than 10–

20 m above the current level [66,67]. All these events could affect

the most recent speciation events and possible inter-basin

connection between the Gulf species of Cambarellus could allow

for dispersal of some of the today widely distributed taxa along the

coastal drainages.

As a lentic-habitat dweller is the widespread way of life for the

subfamily, it is reasonable to think that this could be the same

situation for the ancestors of the different groups. In this case,

formation of Paleolakes during the Middle Miocene (,10.8 MYA)

along the Northern Central Plateau of Mexico and South-East

United States could be important features driving early cladogen-

esis in the Mexican Group.

The pattern of distribution observed in Cambarellus agrees with

those proposed for other freshwater organisms, like the Plateau

Track and western Mountain Track [64]. To explain similar

distributions in fish genera such as Ictalurus, Moxostoma, and

Micropterus, these patterns suggest former hydrographic exchanges

across the present arid plateau. Based on faunal composition and

the finding of sister taxa between those regions like Tampichthys/

Codoma and Algansea/Agosia sister pairs [68], possible connec-

tions between drainages of the South Western Gulf Slope (Nueces,

Colorado and Guadalupe rivers) and those from the northern Rıo

Grande tributaries have been suggested [69]. These connections

could explain the presence of the Northern Central Plateau species

(C. alvarezi, C. areolatus and C. chihuahuae), especially joined to lake

habitats. Extensive lakes associated with the past Rıo Grande

inflow have been documented to cover much of north-western

Chihuahua and southern New Mexico in Pleistocene times, like

the Lake Cabeza de Vaca [70].

The reduction in volume of lacustrine habitats in the Central

Plateau by climatic events may have resulted in a high rate of late

Cenozoic extinction [64], and the patchy distribution pattern of

Cambarellus in this region. This high rate of desiccation, now

increased by human activities [51], could have eroded diversity in

this region, driving to extinction most of the Cambarellus

populations in the Northern Central Plateau of Mexico and could

also explain the current absence of Cambarellus from the rivers

south of the West Gulf Coast drainages. Partial extirpation from a

formerly continuous range due to increasing dry rate during the

Tertiary, has also been seen in different fish groups with a similar

range, like Goodeidae and Cyprinodontidae [71]. Additionally

and continuing southward, former connections between Northern

and Western Central Plateau rivers could explain the presence of

C. occidentalis in the Lower Santiago basin and from there a

connection to the rest of the TMVB could be inferred.

Along the TMVB, the Lerma-Santiago river system is the main

drainage. Previous connections between the Lerma River and

northeastern and western drainages have been suggested for

Goodeidae and Cyprinid fish [71]. Similar to what has been

postulated for freshwater fish groups like the families Atherinidae,

Goodeidae Cyprinidea, diversification in Cambarellus along the

TMVB could have been related to an ancient and successive

fragmentation of the Lerma-Santiago drainage across extensive

lacustrine systems from the Miocene to Pleistocene [71,72].

Separation of the main clades of Cambarellus in the TMVB is

dated along the late Miocene and Pliocene (10.8–4.6 MYA), a

period of high geological activity in Mexico [73]. Formation of the

TMVB advanced in a West-East direction [74], and this could

influence the separation of clades from the main groups of

Cambarellus. This formation could have begun before the presence

of Cambarellus in TMVB, given its absence on the Pacific basins

south to the Zapotlan basin, at its western margin. Major

diversification of the genus took place in an interval of time of

less than 9 MYA.

This study found evidence consistent with a long and complex

evolutionary history of the Cambarellinae. The group’s distribu-

tion has been modified extensively by geologic and climatic

factors. Although there appear to be contrasting causes for

cladogenesis between the two groups, they have similar diversi-

fication rates. In addition, these results showed that genital and

morphological changes widely used in the subfamily in particular

and in crayfish in general, should be compared with other kinds of

evidence in order to make more robust use of morphological

differences for evolutionary inferences.



Supporting Information

Table S1 Voucher numbers and localities of the individuals

analyzed. Proposed new taxa are indicated as Cambarellus sp., and

correspond to the terminal clades indicated with roman numerals

in phylogeny (see Figure 3).

(DOCX)

Acknowledgments

We wish to acknowledge the relevant support in lab work from Rebecca

Scholl (BYU). Museum samples and access to specimens were kindly

provided by Rafael Lemaitre and Karen Reed from US NMNH and

Gonzalo Giribet from MCZ. Valuable help during field trips and with

editing was provided by Patricia Ornelas Garcıa. We thank Daniel P.

Johnson and Paul E. Moler for collection help and for providing some

Cambarellus samples from the US Gulf Coast. Also, some important aspects

of the manuscript were improved from fruitful discussions with Patricia

Ornelas and Mario Garcıa and from the comments kindly made by Joe

Castresana and two anonymous reviewers. Several samples from Mexico

were provided by Fernando Alvarez, Jose Luis Villalobos, Omar

Domınguez and Rodolfo Perez. Very useful suggestions on analyses were

provided by Regina Cunha and Carina Cunha.

Author Contributions

Conceived and designed the experiments: CPL ID KAC. Performed the

experiments: CPL JWB. Analyzed the data: CPL. Contributed reagents/

materials/analysis tools: CPL ID KAC. Wrote the paper: CPL ID KAC

JWB.

References

1. Hobbs HHJ (1974) A checklist of the North and Middle American crayfishes

(Decapoda: Astacidae and Cambaridae). Smithsonian Contributions to Zoology

166: iii+161, 161–294.

2. Fitzpatrick Jr JF (1983) A Revision of the Dwarf Crawfishes (Cambaridae,

Cambarellinae). Journal of Crustacean Biology: 266–277.

3. Hobbs HHJ (1950) A new crayfish of the genus Cambarellus from Texas

(Decapoda, Astacidae). Proceedings of the Biological Society of Washington 63:

89–96.

4. Hobbs H (1989) An Illustrated Checklist of the American Crayfishes (Decapoda,

Astacidae, Cambaridae, Parastacidae). Smithsonian Contributions to Zoology

480: 1–236.

5. Villalobos A (1955) Cambarinos de la fauna Mexicana (Crustacea:Decapoda).

Universidad Nacional Autonoma de Mexico. 290 p.

6. Laguarda A (1961) Contribucion al estudio comparativo de la formula branquial

en la Familia Astacidae (Crustacea:Decapoda): Universidad Nacional Autonoma

de Mexico. 1–74 p.

7. Rojas Y, Alvarez F, Villalobos JL (2002) Morphological variation in the crayfish

Cambarellus (Cambarellus) montezumae (Decapoda:Cambaridae). In: Escobar E,

Alvarez F, editors. Modern approaches to the study of Crustacea. New York:

Kluwer Academic/Plenum Publishers. 311–317.

8. Barraclough T, Vogler A (2000) Detecting the geographical pattern of speciation

from species level phylogenies. Am Nat 155: 419–434.

9. Harvey P, May R, Nee S (1994) Phylogenies without fossils. Evolution 48: 523–

529.

10. Nee S (2006) Birth-Death Models In Macroevolution. Annual Review of

Ecology, Evolution and Systematics 37: 1–17.

11. Rabosky D (2007) Likelihood methods for detecting temporal shifts in

diversification rates. Evolution 60: 1152–1164.

12. Hobbs HHJ (1974) Synopsis of the families and genera of crayfishes

(Crustacea:Decapoda). Smithsonian Contributions to Zoology 164: 1–32.

13. Toon A, Finley M, Staples J, Crandall KA (2009) Decapod Phylogenetics and

Molecular Evolution. In: Martin JW, Crandall KA, Felder DL, editors. Decapod

Crustacean Phylogenetics. Boca Raton, FL: Taylor and Francis Group, LLC.

14. Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy

and high throughput. Nucl Acids Res 32: 1792–1797.

15. Buhay JE (2009) ‘‘COI-like’’ Sequences are Becoming Problematic in Molecular

Systematic and DNA Barcoding Studies. Journal of Crustacean Biology 29: 96–

110.

16. Song H, Buhay JE, Whiting MF, Crandall KA (2008) Many species in one: DNA

barcoding overestimates the number of species when nuclear mitochondrial

pseudogenes are coamplified. Proceedings of the National Academy of Sciences

105: 13486–13491.

17. Swofford DL (1998) PAUP*: Phylogenetic Analysis Using Parsimony (*and

Other Methods). Sunderland, Massachussetts: Sinauer Associates.

18. Akaike H (1974) A new look at the statistical model identification. IEEE

Transactions on Automatic Control 19: 716–723.

19. Schwarz G (1978) Estimating the dimensions of a model. Ann Stat 6 461–464.

20. Posada D (2008) j Model Test: phylogenetic model averaging. Molecular Biology

and Evolution 25: 1253.

21. Guindon S, Gascuel O (2003) A simple, fast, and accurate algorithm to estimate

large phylogenies by maximum likelihood. Systematic biology 52: 696.

22. Felsenstein J (1985) Confidence-Limits on Phylogenies - an Approach Using the

Bootstrap. Evolution 39: 783–791.

23. Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference

under mixed models. Bioinformatics 19: 1572.

24. Huelsenbeck JP, Ronquist F (2005) Bayesian analysis of molecular evolution

using MrBayes. Statistical methods in molecular evolution New York: Springer:

183–232.

25. Rambaut A, Drummond AJ (2007) Tracer v1.4, Available: http://beast.bio.ed.

ac.uk/Tracer.Accesed 2011 Jun 3.

26. Shimodaira H, Hasegawa M (1999) Multiple comparisons of log-likelihoods with

applications to phylogenetic inference. Molecular Biology and Evolution 16:1114–1116.

27. Shimodaira H (2002) An approximately unbiased test of phylogenetic treeselection. Systematic Biology 51: 492.

28. Jobb G (2008) TREEFINDER. Version of October 2008. Munich, Germany.

29. Drummond AJ, Ho SYW, Phillips MJ, Rambaut A (2006) Relaxed

phylogenetics and dating with confidence. PLoS Biology 4: 699–710.

30. Drummond A, Rambaut A (2007) BEAST: Bayesian evolutionary analysis by

sampling trees. BMC Evolutionary Biology 7: 214.

31. Breinholt J, Perez-Losada M., Crandall KA (2009) The timing of thediversification of the Freshwater Crayfishes. In: Martin JW, Crandall KA,

Felder DL, editors. Decapod Crustacean Phylogenetics. Boca Raton, FL: Taylorand Francis Group, LLC. 343–356.

32. Porter M, Perez-Losada M, Crandall K (2005) Model-based multi-locusestimation of decapod phylogeny and divergence times. Molecular Phylogenetics

and Evolution 37: 355–369.

33. Cunningham C, Blackstone N, Buss L (1992) Evolution of king crabs from

hermit crab ancestors.

34. Stillman J, Reeb C (2001) Molecular phylogeny of eastern Pacific porcelain

crabs, genera Petrolisthes and Pachycheles, based on the mtDNA 16S rDNA

sequence: phylogeographic and systematic implications. Molecular Phyloge-netics and Evolution 19: 236–245.

35. Cook BD, Pringle CM, Hughes JM (2008) Phylogeography of an IslandEndemic, the Puerto Rican Freshwater Crab (Epilobocera sinuatifrons). J Hered:

esm126.

36. Knowlton N, Weigt LA (1998) New dates and new rates for divergence across

the Isthmus of Panama. Proceedings of the Royal Society of London Series B-

Biological Sciences 265: 2257–2263.

37. Knowlton N, Weigt LA, Solorzano LA, Mills DK, Bermingham E (1993)

Divergence in proteins, mitochondrial DNA, and reproductive compatibilityacross the Isthmus of Panama. Science 260: 1629.

38. Ferrari L, Lopez-Martinez M, Aguirre-Diaz G, Carrasco-Nunez G (1999)Space-time patterns of Cenozoic arc volcanism in central Mexico: From the

Sierra Madre Occidental to the Mexican Volcanic Belt. Geology 27: 303–306.

39. Feldmann R, Grande L, Birkhimer C, Hannibal J, McCoy D (1981) Decapod

fauna of the Green River formation (Eocene) of Wyoming. Journal ofPaleontology 55: 788–799.

40. Rabosky DL (2007) Likelihood methods for detecting temporal shifts indiversification rates. Evolution 60: 1152–1164.

41. Rabosky D (2006) LASER: a maximum likelihood toolkit for detecting temporal

shifts in diversification rates from molecular phylogenies.

42. Rambaut A, Charleston M (2002) TreeEdit: phylogenetic tree editor, ver. 1.0

alpha 10. Department of Zoology, University of Oxford, Oxford.

43. Yule GU (1925) A Mathematical Theory of Evolution, Based on the Conclusions

of Dr. J. C. Willis, F.R.S. Philosophical Transactions of the Royal Society ofLondon Series B, Containing Papers of a Biological Character 213: 21–87.

44. Pereira SL, Baker AJ, Wajntal A (2002) Combined nuclear and mitochondrialDNA sequences resolve generic relationships within the Cracidae (Galliformes,

Aves). Systematic Biology 51: 946–958.

45. San Mauro D, Gower DJ, Massingham T, Wilkinson M, Zardoya R, et al.

(2009) Experimental design in caecilian systematics: Phylogenetic information of

mitochondrial genomes and nuclear rag1. Systematic Biology 58: 425–438.

46. Fisher-Reid MC, Wiens J (2011) What are the consequences of combining

nuclear and mitochondrial data for phylogenetic analysis? Lessons fromPlethodon salamanders and 13 other vertebrate clades. BMC Evolutionary

Biology 11: 300.

47. Wiens JJ, Kuczynski CA, Smith SA, Mulcahy DG, Sites Jr JW, et al. (2008)

Branch lengths, support, and congruence: testing the phylogenomic approachwith 20 nuclear loci in snakes. Systematic Biology 57: 420–431.

48. Kozak KH, Weisrock DW, Larson A (2006) Rapid lineage accumulation in anon-adaptive radiation: phylogenetic analysis of diversification rates in eastern



North American woodland salamanders (Plethodontidae: Plethodon). Proceedings

of the Royal Society B: Biological Sciences 273: 539–546.

49. Ricklefs RE, Losos JB, Townsend TM (2007) Evolutionary diversification of

clades of squamate reptiles. Journal of Evolutionary Biology 20: 1751–1762.

50. Slowinski JB (2001) Molecular Polytomies. Molecular Phylogenetics and

Evolution 19: 114–120.

51. Contreras-Balderas S, Lozano-Vilano M (1996) Extinction of most Sandıa and

Potosı valleys(Nuevo Leon, Mexico) endemic pupfishes, crayfishes and snails.

Ichthyological exploration of freshwaters Munchen 7: 33–40.

52. Rodrıguez-Almaraz G, Campos E (1994) Distribution and status of the

crayfishes (Cambaridae) of Nuevo Leon, Mexico. Journal of Crustacean Biology

14: 729–735.

53. Sites Jr JW, Marshall JC (2004) Operational criteria for delimiting species.

Annual Review of Ecology, Evolution, and Systematics: 199–227.

54. De Queiroz K (2007) Species concepts and species delimitation. Systematic

Biology 56: 879–886.

55. Perry W, Feder J, Dwyer G, Lodge D (2001) Hybrid zone dynamics and species

replacement between Orconectes crayfishes in a northern Wisconsin lake.

Evolution 55: 1153–1166.

56. Crandall KA, Bininda-Emonds ORP, Mace GM, Wayne RK (2000)

Considering evolutionary processes in conservation biology. Trends in Ecology

& Evolution 15: 290–295.

57. Villalobos A (1943) Estudios de los cambarinos mexicanos, I: Observaciones

sobre Cambarellus montezumae (Saussure) y algunas de sus formas con descripcion

de una subspecie nueva. Annales del Instituto de Biologia 14(2): 587–611: 587–

611.

58. Mateos M, Sanjur OI, Vrijenhoek RC (2002) Historical biogeography of the

livebearing fish genus Poeciliopsis (Poeciliidae : Cyprinodontiformes). Evolution

56: 972–984.

59. Ornelas-Garcıa CP, Dominguez-Dominguez O, Doadrio I (2008) Evolutionary

history of the fish genus Astyanax Baird & Girard (1854) (Actinopterygii,

Characidae) in Mesoamerica reveals multiple morphological homoplasies. BMC

Evol Biol 8: 340.

60. Conner J, Suttkus R (1986) Zoogeography of freshwater fishes of the western

Gulf Slope of North America. The zoogeography of North American freshwater

fishes: New York, John Wiley and Sons: 413–456.

61. McKinney M, McNamara K, Carter B, Donovan S (1992) Evolution of

Paleogene echinoids: a global and regional view. Eocene-Oligocene climatic and

biotic evolution: 349–367.

62. Thomas E (1992) Middle Eocene–late Oligocene bathyal benthic foraminifera

(Weddell Sea): Faunal changes and implications for ocean circulation. Eocene-

Oligocene climatic and biotic evolution Princeton Univ Press, Princeton, NJ:

245–271.

63. Crandall KA, Templeton AR (1999) The Zoogeography and Centers of Origin

of the Crayfish Subgenus Procericambarus (Decapoda: Cambaridae). Evolution 53:

123–134.

64. Miller RR, Smith ML (1986) Origin and geography of the fishes of Central

Mexico. In: Hocutt CH, Wiley EO, editors. The Zoogeography of NorthAmerican freshwater fishes. New York, USA: Wiley-Intersciences publication.

65. Haq BU, Hardenbol J, Vail PR (1987) Chronology of Fluctuating Sea Levels

since the Triassic. Science 235: 1156–1167.66. Riggs S (1984) Paleoceanographic model of Neogene phosphorite deposition,

US Atlantic continental margin. Science 223: 123.67. Swift C, Gilbert C, Bortone S, Burgess G, Yerger R (1986) Zoogeography of the

freshwater fishes of the southeastern United States: Savannah River to Lake

Pontchartrain. In: Hocutt CH, Wiley EO, editors. Zoogeography of NorthAmerican freshwater fishes. New York: John Wiley and Sons. 213–255.

68. Schonhuth S, Doadrio I, Dominguez-Dominguez O, Hillis D, Mayden R (2008)Molecular evolution of southern North American Cyprinidae (Actinopterygii),

with the description of the new genus Tampichthys from central Mexico.Molecular Phylogenetics and Evolution 47: 729–756.

69. Smith M, Miller R (1986) The evolution of the Rio Grande Basin as inferred

from its fish fauna, p. 457–485. In: Hocutt C, Wiley E, editors. TheZoogeography of North American Freshwater Fishes. New York: John Wiley

and Sons.70. Burrows R (1910) Geology of northern Mexico. Boletın de la Sociedad

Geologica Mexicana 7: 85–103.

71. Doadrio I, Domınguez O (2004) Phylogenetic relationship within the fish familyGoodeidae based on cytochrome b sequence data. Molecular Phylogenetics and

Evolution 31: 416–430.72. Barbour CD (1973) A biogeographical history of Chirostoma (Pisces:Atherinidae):

a species flock from the Mexican Plateau. Copeia 1973: 553–556.73. Ferrari L, Conticelli S, Vaggelli G, Petrone CM, Manetti P (2000) Late Miocene

volcanism and intra-arc tectonics during the early development of the Trans-

Mexican Volcanic Belt. Tectonophysics 318: 161–185.74. Ferrari L (2000) Avances en el conocimiento de la Faja Volcanica

Transmexicana durante la ultima decada. Boletın de la Sociedad GeologicaMexicana 53: 84–92.

75. Taylor CA, Hardman M (2002) Phylogenetics of the crayfish subgenus

Crockerinus, genus Orconectes (Decapoda: Cambaridae), based on cytochromeoxidase I. Journal of Crustacean Biology 22: 874–881.

76. Crandall KA, Fitzpatrick Jr JF (1996) Crayfish molecular systematics: using acombination of procedures to estimate phylogeny. Systematic Biology 45: 1.

77. Mokady O, Loya Y, Achituv Y, Geffen E, Graur D, et al. (1999) SpeciationVersus Phenotypic Plasticity in Coral Inhabiting Barnacles: Darwin’s Observa-

tions in an Ecological Context. Journal of Molecular Evolution 49: 367–375.

78. Whiting MF (2002) Mecoptera is paraphyletic: multiple genes and phylogeny ofMecoptera and Siphonaptera. Zoologica Scripta 31: 93–104.

79. Whiting MF, Carpenter JC, Wheeler QD, Wheeler WC (1997) The StrepsipteraProblem: Phylogeny of the Holometabolous Insect Orders Inferred from 18S

and 28S Ribosomal DNA Sequences and Morphology. Syst Biol 46: 1–68.

80. Colgan DJ, McLauchlan A, Wilson GDF, Livingston SP, Edgecombe GD, et al.(1998) Histone H3 and U2 snRNA DNA sequences and arthropod molecular

evolution. Aust J Zool 46: 419–437.



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